Prolonged exposure to nicotine causes profound alterations in nicotine receptors (nAChR), including upregulation and changes in functional status. While in vitro studies show clear differences in these responses among different nAChR subtypes, little is known about differential subtype responses after chronic nicotine exposure in vivo, such as occurs with use of tobacco products or nicotine replacement therapy. If the numbers and activity of some nAChR subtypes is altered more than other subtypes, this would change the pharmacological spectrum of activity and behavioral responses to nicotine; therefore knowledge of subtype-selective responses is essential for understanding nicotine neurobiology. The overarching goal of this proposal is to determine the consequences of chronic in vivo nicotine exposure on expression and function of nAChR subtypes, including alpha4-beta2, alpha-beta4 and alpha6/alpha3-beta2. Chronic drug exposure will be achieved by perfusion of rats with osmotic mini-pumps; it will be compared to acute exposure by injection. Aim 1 will employ a new method of quantitative autoradiography developed in our lab, using [1251] epibatidinewith subtype-selective competitors, that allows measurement of receptor subtypes in small regions, to assess effects of chronic nicotine exposure and recovery from such exposure. We predict that alpha4-beta2 receptors will be much more sensitive to upregulation, and recover more slowly. We will also test the effects of acute nicotine, to test whether effects seen are due to length of time of exposure. We will then use both homogenate binding and autoradiography with more selective radioligands to confirm any changes detected with [125l] epibatidine autoradiography. Aim 2 will use quantitative immunochemical techniques with subunit-selective antibodies to provide important confirmation and extension of any binding changes detected in Aim 1. Aims 3 and 4 will use two different methods to correlate binding changes to functional activity of nAChR subtypes; as above, we hypothesize that activity of alpha4-beta 2 receptors will be disproportionately affected by chronic nicotine. Functional changes caused by chronic (and acute) nicotine and recovery will be directly determined in multiple brain regions by measuring 86Rb efflux (Aim 3). Functional effects of treatments at the systems level will be assessed by measuring nicotinic-stimulated release of different neurotransmitters in selected regions (Aim 4). For these two aims, subtype selectivity will be obtained by the use of selective agonists and antagonists. The systematic application of these different methods to measure numbers and function of nAChR subtypes will allow us to develop a much more complete picture of the brain's response to chronic stimulation by this important drug of abuse.